Project Summary: Nucleosomes are the fundamental units of chromatin, comprised of DNA wrapped around an octamer of histone subunits (H2A, H2B, H3, and H4). Chromatin regulation is achieved through the addition of reversible post-translational modifications (PTMs) to specific histone residues. These PTMs or ?epigenetic modifications? function in systems to dramatically alter downstream gene expression, a hypothesis termed the ?histone code?. Abnormalities in the epigenetic landscape are associated with a collection of age-related diseases and cellular dysfunctions including metastatic cancers and tissue degeneration. Deciphering the histone code toward the development of therapeutics is thus, dependent on nucleosome-based reagents to model how PTM combinations alter epigenetic signaling. EpiCypher is pioneering the development of recombinant nucleosomes carrying specific PTMs (designer nucleosomes [dNucs]) for drug discovery applications. dNuc-based reagents however, are limited to the production of nucleosomes carrying the same modification in a given histone subunit dimer. For example, dNucs carrying a histone H3 subunit trimethylated at lysine 4 (H3K4me3) will have both H3 subunits modified. Recent studies show that nearly half of histone PTMs are added asymmetrically in the genome, revealing a striking (and previously unappreciated) additional level of PTM complexity. ?bivalent? promoters, containing both active (H3K4me3, H3K36me2/3) and repressive (H3K27me3) marks, are also asymmetrically modified at the nucleosome level. This epigenetic signature is observed in stem cells / cancer cells and plays key roles in stem cell maintenance / differentiation. Significantly, PTM symmetry exerts a pronounced effect on the activity of chromatin modifying enzymes. For instance, the activity of EZH2 (a histone H3K27-specific methyltransferase associated with several cancers [e.g. leukemia] and adult stem cell senescence) is dramatically enhanced by asymmetric vs. symmetric H3K4me3. These findings suggest that PTM symmetry provides an additional regulatory layer to the histone code, thereby altering epigenetic signaling by preferentially recruiting specific chromatin modifying proteins. Currently, there are no available tools to examine the function of epigenetic regulators in the context of asymmetric PTMs. In this proposal, we will optimize novel methods to generate asymmetrically modified dNucs at commercial-grade and -scale (Aim 1) and develop an innovative drug discovery platform that utilizes these physiological nucleosomes as biochemical substrates to recapitulate in vivo activity of chromatin regulators at H3K4me3/H3K27me3 bivalent promoters (Aim 2). In Phase II, we will 1) scale up manufacturing of asymmetrically modified dNucs for our industry/pharmaceutical partners and 2) develop a portfolio of high- throughput assays (biochemical and cell-based) for drug discovery and development using other disease- relevant asymmetric PTMs, including H3K27me3/H3K27ac (lung cancer) and H4K20me1 (leukemia, lung and pancreatic cancer).